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Indo American Journal of Pharmaceutical Sciences (IAJPS) is an international, peer-reviewed, multidisciplinary monthly journal, devoted to pharmaceutical sciences. The aim of IAJPS is to increase the impact of pharmaceutical research both in academia and industry, with strong emphasis on quality and originality. IAJPS publishes Original Research Articles, Short Communications, Review Articles in all areas of pharmaceutical sciences from the discovery of a drug up to clinical evaluation.Topics covered are: Pharmaceutics, Bio Pharmaceutics and Pharmacokinetics, Novel Drug Delivery System, Pharmaceutical chemistry including medicinal and analytical chemistry; Pharmacognosy including herbal products standardization and Phytochemistry; Pharmacology, Bio Technology: Allied sciences including drug regulatory affairs, Pharmaceutical Marketing, Pharmaceutical Microbiology, Pharmaceutical biochemistry, Pharmaceutical Education and Hospital Pharmacy.
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IAJPS 2015, Volume2 (5), 947-954 Manoj Lamichhane et al ISSN 2349-7750
w w w . i a j p s . c o m
Page 947
ISSN 2349-7750
IINNDDOO AAMMEERRIICCAANN JJOOUURRNNAALL OOFF
PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS
Available online at: http://www.iajps.com Research Article
DEVELOPMENT AND VALIDATION OF RP-UFLC AND UV
SPECTROPHOTOMETRIC METHODS FOR DETERMINATION OF
10-(4'-N-[(-HYDROXYETHYL) PIPERAZINE] BUTYL)-1, 3-DIMETHYL-10H-ACRIDINONE IN BULK DRUG AS A POTENT DNA
INTERCALATOR Manoj Lamichhane1*, N.K. Sathish1, Ankit Acharya2, Braj Kishor Yadav1,
Saroj Paudel3 1Department of Pharmaceutical Analysis, Sri Adichunchanagiri College of Pharmacy,
B.G. Nagara-571 448, Mandya Dist. Karnataka, India 2Department of Pharmaceutical Pharmaceutics, Sri Adichunchanagiri College of Pharmacy,
B.G. Nagara-571 448, Mandya Dist. Karnataka, India 3Department of Pharmacy practice, Sri Adichunchanagiri College of Pharmacy,
B.G. Nagara-571 448, Mandya Dist. Karnataka, India
Abstract: The present RP-UFLC and UV methods are relatively simple, rapid, robust and highly precise in the
determination of 10-(4'-N-[(-Hydroxyethyl) piperazine] butyl)-1,3-dimethyl-10H-acridinone (HBA) in bulk drug. The chromatographic separation of the drug was achieved on eclipse plus C8 column (2504.6mm i.d.
5m) using a mobile phase of methanol: water (50:50). The flow rate was 1.0 ml/min and effluents were
monitored at 267 nm. The separation was achieved within 3.0 0.2 min. The method showed good linearity in
the range of 0-10 g/mL with coefficient of correlation 0.9906. The intra and inter day RSD ranged within
limits. The limit of detection and limit of quantification were 0.140 and 0.424 g/mL, respectively. UV
spectrophotometric determination of HBA in bulk drug was done by using 0.1N HCL as solvent and the
absorption maxima was found to be 262 nm. A linear response was observed in the range of 0-18 g/mL with a
correlation coefficient of 0.999. The method was validated as per ICH guidelines and RSD ranged within limits.
These methods can be successfully employed to quantify HBA in the bulk drug and can be used for routine
quality control purposes.
Keywords: 10-(4'-N-[(-Hydroxyethyl) piperazine] butyl)-1,3-dimethyl-10H-acridinone (HBA), Method Validation, RP-UFLC, UV spectrophotometry.
corresponding author:
Manoj Lamichhane
Department of Pharmaceutical Analysis,
Sri Adichunchanagiri College of Pharmacy,
B.G. Nagara-571 448, Karnataka, India.
Please cite this article in press as Manoj Lamichhane et al,, Development And Validation Of RP-UFLC And
UV Spectrophotometric Methods For Determination Of 10-(4'-N-[(-Hydroxyethyl) Piperazine] Butyl)-1, 3-Dimethyl-10h-Acridinone In Bulk Drug As A Potent DNA Intercalator, Indo American J of Pharm Sci,
2015;2(5):947-954.
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IAJPS 2015, Volume2 (5), 947-954 Manoj Lamichhane et al ISSN 2349-7750
w w w . i a j p s . c o m
Page 948
INTRODUCTION:
HBA is chemically 10-(4'-N-[(-Hydroxyethyl) piperazine] butyl)-1,3-dimethyl-10H-acridinone,
which is an 1,3-dimethyl acridone derivative
having good cytotoxic activity and DNA
intercalation properties. Various 9-acridone
derivatives with or without an alkyl side chain
attached to the N-position were found to exhibit
anticancer and antibacterial activities. HBA is
freely soluble in methanol, water, DMSO and 0.1N
HCL. HBA showed higher DNA-binding constant
(Ki) 10.32 x 10 x M-1 and also showed relatively
better cytotoxic activity against tumor cell lines,
which may be attributed, at least in part, to its
intercalative association and high binding afnity with the DNA. As for the structure-activity
relationship, HBA bearing the planar tricyclic ring
with butyl (-hydroxyethyl) piperazine side chain shows stronger binding property [1]. HBA
exhibited potent cytotoxic activity against MCF-7
cell line, HL-60 and Ehrlich Ascites Carcinoma
(EAC) cell line (Satish et al., 2010). The Structural
Elucidation of synthesized HBA was confirmed by 1H-NMR, 13C-NMR, mass spectroscopy and
elemental analysis [1].
N
O CH3
CH3
(H2C)4 N
N CH2CH2OH Structure of HBA As far there are very few numbers of analytical
methods reported in the literature for the
determination of 1,3-dimethyl acridone derivatives.
Officially assay of HBA is not described in any
pharmacopoeias. From literature survey, there has
been no HPLC and UV spectroscopy method
reported for the estimation of HBA in bulk drug.
In the present work, an attempt was made to
provide a newer, simple, low cost, robust, precise
and reliable UFLC and UV-spectrophotometric
methods for the effective quantitative
determination of HBA as an active pharmaceutical
ingredient. The developed method can be
successfully applied in estimation of HBA. The
results of analysis were validated as per
International Conference on Harmonization
guidelines [2].
MATERIALS AND METHODS:
Instrumentation and Analytical conditions The RP-UFLC method was performed by using
Shimadzu Prominence binary gradient, UFLC
instrument. The instrument was provided with a
reversed-phase C-8 (250 mm X 4.6 mm, 5m i.d.),
a binary prominence LC 20AD pump and an SPD
M20A prominence diode array detector. A 20 L
Hamilton injection syringe was used for sample
injection. LC solutions software was used for Data
acquisition. HPLC grade methanol (Rankem pvt.
Ltd.) and Milli-pore water were used in the study.
A freshly prepared binary mixture of methanol:
water (50:50 %v/v) was used as the mobile phase.
The mobile phase was filtered through a 0.45
membrane filter and degassed before use. The flow
rate of mobile phase was maintained at 1.0
mL/min. The detection of the drug was carried out
at 267 nm. The analysis was carried out at 18C.
The UV method was performed on a Shimadzu
UV-Visible Spectrophotometer 1700, and a pair of
1-cm matched quartz cells were used to measure
absorbance of the resulting solution. In this UV
method solvent used is 0.1N Hydrochloric acid (S d
fine-chem limited) and double distilled water is
used as diluents. Maximum absorbance was
observed at 262 nm [3].
Drug sample The drug sample HBA was synthesized and
purified by column chromatography and its purity
is checked by RP-UFLC.
Preparation of stock and working standard
solutions
RP-UFLC Method Approximately 100 mg of HBA was accurately
weighed and transferred to a 100 ml volumetric
flask and dissolved with minimum quantity of
mobile phase. The flask was sonicated for 10 min
to dissolve the solids. It was diluted to volume with
mobile phase and mixed well, which yields
standard stock solution of concentration 1000g/ml
(stock-1) of HBA. Pipette out 2ml from stock-1 and
dissolved in 100 ml of mobile phase to get working
standard stock solution of 20g/ml (stock-2) of
HBA.
UV Method Approximately 40mg of HBA was accurately
weighed and transferred into a 100mL volumetric
flask and diluted to volume with 0.1N HCL which
gave 400g/ml (stock-1) concentration of HBA.
Pipette out 5 ml from stock-1 and transferred into
100 ml volumetric flask and diluted with 0.1N
HCL which yields working standard concentration
of 20g/ml (stock-2) of HBA.
Linearity and Construction of Calibration
Curve
RP-UFLC Method The quantitative determination of the drug was
accomplished by using methanol: water (50:50 v/v).
The column was equilibrated with the mobile phase
for at least 30 minutes prior to the injection of the
drug solution. The Linearity (Beers law) of detector response was established by plotting a
graph of concentration versus peak area of HBA
standard and determining the correlation
coefficient. A series of solution of HBA standard
solution in the concentration ranging from about 0-
10 g/mL were prepared and injected into the
UFLC system (figure 1). Each dilution was
injected six times into the column. The drug
analyte was monitored at 267 nm and the
IAJPS 2015, Volume2 (5), 947-954 Manoj Lamichhane et al ISSN 2349-7750
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Page 949
corresponding chromatograms were obtained. From
these chromatograms, a plot of concentrations over
the peak areas was constructed. A representative
chromatogram for the separation of HBA is given
in figure 2. Linearity data of HBA by RP-UFLC
method is depicited in table 1.
y = 24847x + 7210.R = 0.990
0
50000
100000
150000
200000
250000
300000
0 5 10 15
pe
ak a
rea
concentration g/ml
linearity curve of HBA by RP-UFLC
area
Linear (area)
Figure 1: The linearity curve of HBA by RP-UFLC
method
Figure 2: Chromatogram of HBA
Table 1: linearity data of HBA by RP-UFLC method
Sl. No. Concentration (g/ml)
Retention time
(min)
Peak area
1. 0 0 0
2. 2 3.210 58328
3. 4 3.212 105887
4. 6 3.218 170905
5. 8 3.210 209254
6. 10 3.215 244299
HBA is 10-(4'-N-[(-Hydroxyethyl) piperazine] butyl)-1,3-dimethyl-10H-acridinone.
IAJPS 2015, Volume2 (5), 947-954 Manoj Lamichhane et al ISSN 2349-7750
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Page 950
UV Method:
Suitable aliquots of the standard solution of HBA
were taken in 10 mL volumetric flasks. The volume
was then made up to the mark with 0.1N
hydrochloric acid to prepare a series of standard
solutions containing 0-18 g/ml. The UV spectrum
for HBA is shown in figure 3. Absorbance was
measured at 262 nm against blank. Linearity graph
is shown in figure 4.
Fig 4: UV-Spectrum of HBA
Fig 4: The linearity curve of HBA by UV spectrophotometry method
IAJPS 2015, Volume2 (5), 947-954 Manoj Lamichhane et al ISSN 2349-7750
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Method Validation
HPLC and UV Methods
Precision
The precision of the analytical method was studied
by analysis of multiple sampling of homogeneous
sample. The precision of a method was calculated
by performing intra-day precision and inter-day
precision studies. The average was taken and %
RSD was calculated and the values were within the
limit i.e. (< 2), so the method was found to be
highly precise. The precision data observed were
shown in table 2 and table 3 by RP-UFLC and UV
method respectively.
Linearity
The linearity of the analytical method was its
ability to elicit test results which are directly
proportional to analyte concentration in samples
within a given range. Linearity was studied by
preparing standard solution at five different
concentration levels. The linearity range was found
to be 0-10 g/ml. Twenty L of each solution was
injected into the RP-UFLC system through manual
injector. Peak areas were recorded for all the
chromatogram. The correlation coefficient of HBA
was found to be 0.9906. Peak area of linearity
range and the parameters were calculated and
presented in Table 1. The linearity curve of HBA
was shown in figure 3. The linearity of the
proposed UV spectroscopy method was established
by preparing various aliquots of the standard
solution of the HBA from stock solution and
analyzed. The drug showed linearity in the range of
0-18g/ml with correlation coefficient 0.999.
Linearity data are shown in table 4 and linearity
graph is plotted, concentration in X-axis and
absorbance in Y-axis shown in figure 5. These data
clearly demonstrates that the developed method
have adequate sensitivity to the concentration of
the analyte in the sample.
Table 2: Results of precision of HBA by RP-UFLC method
Concentration (g/ml) Peak area
Statistical Analysis
Mean- 105097
SD- 1054.921
% RSD-1.00376
%RSD- 1.00376
4 104108
4 106309
4 104583
4 106562
4 104604
4 104416
SD is standard deviation for n = 6 observations, %RSD is relative standard deviation.
Table 3: Results of precision of HBA by UV spectrophotometry method
Concentration (g/ml) Absorbance Statistical Analysis
10 0.573 Mean- 0.574
SD- 0.002366
%RSD- 0.41
10 0.572
10 0.572
10 0.577
10 0.577
10 0.573
IAJPS 2015, Volume2 (5), 947-954 Manoj Lamichhane et al ISSN 2349-7750
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Table 4: Linearity results of HBA by UV-
spectrophotometry method
Sl.
No.
Concentration
(g/ml)
Absorbance
1. 0 0
2. 2 0.139
3. 4 0.243
4. 6 0.342
5. 8 0.460
6. 10 0.578
7. 12 0.692
8. 14 0.814
9. 16 0.895
10. 18 1.023
Limit of Detection (LOD) and Limit of
Quantification (LOQ):
Limit of detection (LOD) is the lowest amount of
analyte in the sample that can be detected. Limit of
quantification (LOQ) is the lowest amount of
analyte in the sample that can be quantitatively
determined by suitable precision and accuracy. The
LOD and LOQ were separately determined and
reported, based on the calibration curve of standard
solution. The relative standard deviation of the
regression line or the standard deviation of yintercepts of regression lines may be used to
calculate LOD and LOQ. LOD = 3.3 S/m and
LOQ = 10 S/m, where, S is the standard
deviation of Y-intercepts of regression line and m
is the slope of the calibration curve. The LOD and
LOQ of the developed methods were determined
by analyzing progressively low concentration of
the standard solutions using the developed
methods. The LOD is the lowest concentration of
the analyte that gives a measurable response (signal
to noise ratio of 3.3). LOD of HBA was found to be
0.140 g / ml. The LOQ is the lowest concentration
of the analyte, which gives response that can be
accurately quantified (signal to noise ratio of 10).
The LOQ of HBA was found to be 0.424 g / ml.
Robustness
Robustness of the method was determined by
making slight change in the chromatographic
condition in RP-UFLC method. Robustness is the
ability to provide accurate and precise results under
a variety of conditions. It was observed that there
were no marked changes in the chromatograms.
Results for robustness study were shown in table 5
which indicated that the small change in the
temperature conditions did not significantly affect
the determination of HBA in bulk drug. Analysis
was carried out at two different temperatures, room
temperature and at 18C to determine the
robustness of the method and the respective areas
was measured. The results were indicated as %
RSD. Robustness of the method by UV
spectroscopy was determined by making slight
change in the temperature condition. It was
observed that there were no marked changes in the
absorbance.
Table 5: Results of robustness for HBA by UV spectrophotometry method
Room Temperature Temperature 18 C
Concentration
(g/ml)
Absorbance
Statistical
Analysis
Concentration
(g/ml)
Absorbance
Statistical
Analysis
10 0.573 Mean- 0.5758
SD- 0.002483
%RSD- 0.43
10 0.572
Mean = 0.5708
SD = 0.001169
%RSD = 0.204
10 0.574 10 0.571
10 0.574 10 0.571
10 0.579 10 0.569
10 0.577 10 0.572
10 0.578 10 0.570
IAJPS 2015, Volume2 (5), 947-954 Manoj Lamichhane et al ISSN 2349-7750
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Ruggedness
Ruggedness was determined by carrying out
analysis by two different analysts and the
respective absorbance was noted and the results
were indicated as % RSD. It was observed that
there were no marked changes in the absorbance.
The results for ruggedness study which indicated
that the analysis by different analysts did not
significantly affect the determination of HBA in
bulk drug (table 6).
RESULTS AND DISCUSSION:
RP-UFLC Method
The present study was aimed at developing a
sensitive, simple, precise and robust UFLC method
for the analysis of HBA in bulk drug. In order to
achieve optimum separation of the component
peaks, mixtures of methanol with water and
acetonitrile were tested as mobile phase on C-8
stationary phase. A binary mixture of methanol and
water (50:50 %v/v) was selected, as the
chromatographic peaks were well defined and
resolved with no tailing. The retention time
obtained for HBA was 3.2 min. Each of the
samples was injected six times and the same
retention times were observed in all the cases. The
peak areas of HBA were reproducible as indicated
by low coefficient of variation. A good linear
relationship (r2 = 0.9906) was observed over the
concentration range of 0-10 g/ml of HBA. The
regression curve was constructed by linear
regression and its mathematical expression was y =
24847x + 7210.4 (where y gives peak area and x is
the concentration of the drug). The absence of
additional peaks indicated non-interference of other
impurities in the drug sample is shown in figure
2. The low percentage relative standard deviation
indicated the reproducibility of the assay of HBA
in bulk drug. The deliberate changes in the method
have not much affected the tailing factor, mean
peak area and the retention time. This indicated the
robustness of the method. System suitability
parameters were studied with six replicates
standard solution of the drug and the calculated
parameters are within the acceptance criteria and
the results are shown in table 7.
Table 6: Results of ruggedness for HBA by UV spectrophotometry method
Table 7: Characteristic parameters of HBA for the proposed RP-UFLC method
Parameters RP-UFLC
Linearity range (g / ml) 0-10
Detection wavelength (nm) 267
Mobile phase (Methanol : water) 50 : 50(v / v)
Retention time (min) 3.2
Regression equation (Y) Y = 24847x + 7210.4
Slope (m) 24847
Intercept (c) 7210.4
Correlation coefficient(r2) 0.9906
Precision (% RSD)
Intraday Precision (n=6)
Interday Precision (n=6)
1.00376
0.79
1.66
Limit of detection (g / ml) 0.140
Limit of quantitation (g / ml) 0.424
Theoretical plates 4240.899
Tailing factor (asymmetry factor) 0.940
Analyst 1
Analyst 2
Concentration
(g/ml)
Absorbance Statistical
Analysis
Concentration
(g/ml)
Absorbance Statistical
Analysis
10 0.576
Mean- 0.575
SD- 0.001789
%RSD-
0.311105
10 0.579
Mean = 0.57633
SD = 0.001862
%RSD = 0.323
10 0.574 10 0.578
10 0.575 10 0.576
10 0.574 10 0.576
10 0.573
10 0.578
IAJPS 2015, Volume2 (5), 947-954 Manoj Lamichhane et al ISSN 2349-7750
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Table 8: Characteristic Parameters of HBA for Proposed UV Spectrophotometry Method
Parameters RESULTS
Linearity range (g / ml) 0-18
Detection wavelength (nm) 262
Regression equation (Y) Y = 0.0561x + 0.0135
Slope (m) 0.0561
Intercept (c) 0.0135
Correlation coefficient(r2) 0.9991
Precision (% RSD)
Intra-day Precision (n=6)
Inter-day Precision (n=6)
0.41
0.158
0.364
Limit of detection (g / ml) 0.139
Limit of quantitation (g / ml) 0.421
UV Method
The solubility of HBA was determined in a variety
of solvents. The proposed method is simple and
precise and do not suffer from any interference due
to impurities. Method was validated in terms of
ruggedness, robustness, precision, LOD, LOQ and
linearity. The characteristic parameters of HBA for
proposed UV spectrophotometry method are shown
in table 8.
CONCLUSION: A validated UFLC and UV methods have been
developed for the determination of HBA in bulk
drug. The proposed methods are simple, rapid,
precise, cost-effective and specific. Its
chromatographic run time of 10 minutes allows the
analysis of a large number of samples in a short
period of time. Therefore, it is suitable for the
routine analysis of HBA in bulk drug. The
simplicity of the method allows for application in
laboratories that lack sophisticated analytical
instruments such as LC-MS/MS or GC-MS/MS
that are complicated, costly and time consuming
rather than a simple HPLC and UV methods. The
results for standard drug HBA in the proposed
method were found to be satisfactory. In an over
view the results indicate that the method is precise
enough for the analysis of the drug. Considering
the possible worldwide development of new drug
i.e. HBA as cytotoxic agent and DNA intercalator,
the proposed method could be useful for the quality
control, purity and identification of HBA in bulk
drug in research and development and in
pharmaceutical industries.
ACKNOWLEDGEMENTS:
The authors are thankful to Professor
Dr. B. Ramesh, Principal, Sri Adichunchanagiri
College of Pharmacy, B.G. Nagara-571448,
Karnataka, India for providing required facilities to
carry out this research work.
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Synthesis, chemical characterization of novel
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2. Sathish NK, Raghavendra NM, Mohammed IA, Anti-tumour Activity of 1, 3- Dimethyl
Acridones Against Ehrlich Ascites Carcinoma,
Intl J. Drug D Dis, 2010;1:4:331-35.
3. Chongde S, Xian L, Changjie X, Shanglong Z, Kunsong C, Qingjun C, et al., Determination
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